Cartilage calcification is an essential step in the processes of fracture healing and bone growth (endochondral ossification). The goal of the proposed studies is to determine the ways in which cartilage calcification is regulated. Since mammals and birds demonstrate different patterns of cartilage calcification they provide excellent models for examining these regulatory process. Mammalian and avian cartilage calcification can be differentially modeled in vitro using a well developed model of chondrogenesis. Chondrogenesis (cartilage formation) occurs in vitro when mesenchymal cells (isolated from embryonic limb buds) are cultured at high density in media containing 1 mM calcium: the matrix will calcify in the presence of 3-4 mM inorganic phosphate. The long range goal of the proposed study is to use chick and mouse mesenchymal cell systems to characterize events in initial calcification. Chemical analyses, light and electron microscopy, x-ray diffraction and infra-red spectroscopy, and immunochemistry will be used to evaluate the mineralized matrix formed in vitro. Experiments are designed initially to optimize the culture conditions to provide maximal "physiologic" matrix mineralization by varying a) ascorbic acid, b) phosphate, c) beta-glycerol phosphate, d) calcium, e) fetal calf serum, and f) atmospheric conditions. The calcified tissue formed in the avian and murine cell culture systems will be compared to each other and to mineralized cartilage produced in vivo, with respect to: a) type of matrix undergoing mineralization, b) mineral characteristics, and c) cells regulating the mineralization process. The cultured cells will than be chemically or physically modified in order to test the hypothesis that viable, as opposed to necrotic, cells are responsible for mineralization. The effect of alteration of matrix properties on mineralization will be examined by using agents that alter: a) cartilage differentiation and development (BrDU and vitamin A), b) proteoglycan synthesis, composition and/or properties (vitamin A, xylosides, hyaluronidase, polylysine), and c) synthesis of other extracellular matrix proteins (cAMP). The culture systems will be examined to determine how clinically relevant trace elements (F, A1, Fe, Ga, Ti, V, Ni, Cr and Co) control the extent of cell mediated mineralization. These cell culture studies are unique due to their emphases on the mechanism of cell-mediated calcification. Their clinical significance comes from the basic information on calcification mechanisms they will provide as well as from the specific data they will generate on the effect of certain therapies on these mechanisms.